Identification and cloning of Silybum marianum chalcone synthase gene/gene family

Silymarin is a flavonoid compound derived from milk thistle plant (Silybum marianum) seeds comprising several pharmacological applications. Chalcone synthase (CHS) is a key enzyme in the biosynthesis of flavonoids, thereby identification of CHS gene/ gene family in milk thistle plant can be of great importance. The available sequences of CHS genes from different plants collected and aligned, to detect the conserved and diverged regions. Then, 4 degenerate primers (2 forward primers (F1, F2) and 2 reverse primers (R1, R2)) designed based on the CHS consensus sequences. The fragments of CHS genes amplified from Borazjan's native genotype and Majar's modified genotype by polymerase chain reaction which cloned and sequenced, thereafter. Analysis of the resultant nucleotide and deduced amino acid sequences of F1R2 and F2R1 fragments lead to identification of two different members of CHS gene family from Silybum marianum. F1R2 sequence analysis also revealed that this CHS gene of milk thistle contains two exons separated by an intron (208 bp) which is located at the conserved position. 6 specific primers (3 forward primers and 3 reverse primers) designed based on the diverged regions of each member for amplification of related cDNA from majar's total RNA in the RACE system. Amplified fragment of cDNA in 3'RACE and 5'RACE were cloned and sequenced. Full length cDNA was identified by overlapping the 3'RACE and 5'RACE sequences of member1 whose open reading frame contains 1239 bp including exon 1 (244 bp) and exon 2 (1182 bp) encoding 63 and 349 amino acid residues respectively. Altogether, analysis of the resulting nucleotide and deduced amino acid sequences lead to identification of three different members of chalcone synthase from Silybum marianum containing the conserved chalcone synthase Cterminal and N-terminal domains.